Insights into Intra-Tumoral Heterogeneity: Transcriptional Profiling of Chemoresistant MPM Cell Subpopulations Reveals Involvement of NFkB and DNA Repair Pathways and Contributes a Prognostic Signature

Chemoresistance is a hallmark of malignant pleural mesothelioma (MPM) management and the expression of ALDH1A3 is responsible for the survival and activity of MPM chemoresistant cell subpopulations (ALDH^bright cells). We enriched mesothelioma ALDH^bright cells to near homogeneity by FACS sorting and an Aldefluor assay and performed unbiased Affymetrix gene expression profiling. Viability and ELISA assays were used to rule out significant apoptosis in the sorted cell subpopulations and to assess target engagement by butein. Statistical analysis of the results, pathway enrichment and promoter enrichment were employed for the generation of the data. Q-RTPCR was used to validate a subset of the identified, modulated mRNAs In this work, we started from the observation that the mRNA levels of the ALDH1A3 isoform could prognostically stratify MPM patients. Thus, we purified MPM ALDH^bright cells from NCI-H2595 cells and interrogated their gene expression (GES) profile. We analyzed the GES of the purified cells at both a steady state and upon treatment with butein (a multifunctional tetrahydroxy-chalcone), which abates the ALDH^bright cell number, thereby exerting chemo-sensitizing effects in vitro and in vivo. We identified 924 genes modulated in a statistically significant manner as a function of ALDH status and of the response to the inhibitor. Pathway and promoter enrichment identified the molecular determinant of high ALDH status and how butein treatment altered the molecular portrait of those chemoresistant cell subpopulations. Further, we unraveled an eighteen-gene signature with high prognostic significance for MPM patients, and showed that most of the identified prognostic contributors escaped the analysis of unfractionated samples. This work proves that digging into the unexplored field of intra-tumor heterogeneity (ITH) by working at the cell subpopulation level may provide findings of prognostic relevance, in addition to mechanistic insights into tumor resistance to therapy.     

Effect of prepartum selenium treatment on uterine involution in the dairy cow

Selenium injections and oral vitamin E supplementation prepartum were related to: postpartum uterine involution (decrease in uterine size per unit time) and days to minimum uterine size in a 2 X 2 factorial design. Complete data were analyzed from 64 cows. Groups were selenium plus vitamin E, vitamin E, selenium, and control. Factors significantly affecting uterine size between 14 and 50 d postpartum were cow weight, days postpartum-linear, days postpartum-quadratic, day X metritis, and day X metritis X selenium treatment. Days to minimum uterine size were significantly less in cows with metritis and selenium treated when compared with cows with metritis and not selenium treated (32.9 vs. 35.8).     

Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae

In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.     

Trace element concentrations in the Pacific harbor seal (Phoca vitulina richardii) in central and northern California

To determine concentrations of trace elements (THg, MeHg, Se, and Pb) in tissues of the Pacific harbor seal (Phoca vitulina richardii), live (n=186) and dead seals (n=53) were sampled throughout central and northern California from March 2003 to January 2005. There were significant differences in THg concentrations in blood and hair based on age (p<0.001). Adult male harbor seals had greater THg concentrations in their hair than adult female harbor seals (p<0.003). THg concentrations in liver increased linearly with age and delta15N (p<0.001); whereas, MeHg concentrations in liver increased exponentially until approximately 5 years of age with an asymptote at 1.3 microg/g wet weight. MeHg expressed as a percentage of THg (%MeHg) was best described by a decay function (r2=0.796, p<0.001), decreasing to a minimum at 4 years of age. Hepatic Se increased with age and was in equimolar ratios with THg in adults; whereas, molar ratio of Se:THg in pups deviated from a 1:1 ratio. Significant differences among study locations in THg concentrations in blood and hair were not detected. Assessing the possible effect of sampling location on Hg concentrations, however, was confounded and limited by lack of equal sample sizes for basic age and sex cohorts, a common dilemma in pinniped research.     

Cytotoxicity of polycyclic aromatic hydrocarbon o-quinones in rat and human hepatoma cells

A novel pathway of polycyclic aromatic hydrocarbon (PAH) metabolism involves the oxidation of non-K-region trans-dihydrodiols by dihydrodiol dehydrogenase (DD) to yield PAH o-quinones whose cytotoxicity and genotoxicity are unknown. The cytotoxicity of several PAH o-quinones derived from this reaction [naphthalene-1,2-dione (NPQ), benzo[a]pyrene-7,8-dione (BPQ), and 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBAQ)] was examined in rat (H-4IIe) and human (Hep-G2) hepatoma cells which are known to express DD. 2-Methylnaphthalene-1,4-dione (menadione), a known cytotoxic p-quinone, was used as a positive control. Hepatoma cells (1 x 10(6) cells/mL) were exposed to PAH o-quinones (1-100 microM) for 0-4 h, and cell viability and survival were measured and related to O2.- production and changes in redox potential [GSSG/GSH and NAD(P)+/NAD(P)H]. Three different modes of cytotoxicity were observed: (1) NPQ (no bay region) and DMBAQ (methylated bay region) were as cytotoxic as menadione in reducing cell survival but had less effect on cell viability. These o-quinones adversely affected GSH levels and the redox state of the cell and caused an increase in the production of O2.- in cell suspensions. This cytotoxicity was not enhanced by dicoumarol (10 microM), a DT-diaphorase inhibitor, implying that this enzyme is unable to prevent these PAH o-quinones from entering one-electron redox-cycles. (2) BPQ (bay region only) was the least cytotoxic of the PAH o-quinones studied. BPQ decreased cell viability (< 40% at 20 microM) but did not adversely affect cell survival or the redox state of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation between Melting and Textural Properties of Process Cheddar Cheese

Meltability and textural characteristics were evaluated in 48 batches of process Cheddar cheese prepared in pilot plant equipment. Correlation between melting spread at 139°C and cohesiveness at 21°C was positive and large. Prolongation of cooking up to 15 min at 74°C lowered meltability and cohesiveness. Within the range of weighted average ages of cheese (3 to 5.8 mo), no relation between melting spread or cohesiveness and age was consistent.     

Hydromorphological,hydraulic and ecological effects of restored wood: findings and reflections from an academic partnership approach

Large wood (re)introduction can deliver multiple benefits in river restoration, but there is a dearth of the detailed and longer‐term post‐project monitoring and evaluation required for improving best practice. We present findings from an academic partnership approach to post‐project evaluation, based on successive MSc research projects on restored large wood in the Loddon catchment, UK. Field and modelling data reveal: (i) key differences in large wood features between restored and natural reaches; (ii) increased hydraulic retention and changes to mesohabitats associated with large wood; (iii) differences in macroinvertebrate community composition around large wood but a lack of site‐level effects; (iv) interactions between macrophytes and large wood that may be specific to restored reaches; (v) a need for further field and modelling studies to inform the accurate representation of large wood in hydraulic models. Some key challenges in partnership working are identified to aid planning and effectiveness of future collaborations.